ENZYBIOTICS as Drugs and Therapeutics

Edited by

TOMAS G. VILLA School of Biotechnology University of Santiago de Compostela and PATRICIA VEIGA-CRESPO Department of Microbiology Faculty of Pharmacy University of Santiago de Compostela

A JOHN WILEY & SONS, INC., PUBLICATION

ENZYBIOTICS

ENZYBIOTICS Antibiotic Enzymes as Drugs and Therapeutics

Edited by

TOMAS G. VILLA School of Biotechnology University of Santiago de Compostela and PATRICIA VEIGA-CRESPO Department of Microbiology Faculty of Pharmacy University of Santiago de Compostela

A JOHN WILEY & SONS, INC., PUBLICATION Copyright © 2010 by John Wiley & Sons, Inc. All rights reserved

Published by John Wiley & Sons, Inc., Hoboken, New Jersey Published simultaneously in Canada

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Library of Congress Cataloging-in-Publication Data Enzybiotics : antibiotic enzymes as drugs and therapeutics / [edited by] Tomas G. Villa, Patricia Veiga Crespo. p. ; cm. Includes index. ISBN 978-0-470-37655-3 (cloth) 1. Enzymes–Therapeutic use. 2. Antibacterial agents. I. Gonzalez Villa, Tomas. II. Veiga Crespo, Patricia. [DNLM: 1. Anti-Bacterial Agents–pharmacology. 2. Anti-Bacterial Agents–therapeutic use. 3. –enzymology. 4. Bacteriophages–genetics. 5. Drug Resistance–drug effects. QV 350 E612 2010] RM666.E55E575 2010 615′.35–dc22 2009025908

Printed in the United States of America

10 9 8 7 6 5 4 3 2 1 CONTENTS

PREFACE vii CONTRIBUTORS ix

1 ENZYBIOTICS AND THEIR POTENTIAL APPLICATIONS IN MEDICINE 1 Jan Borysowski and Andrzej Górski

2 ADVANTAGES AND DISADVANTAGES IN THE USE OF OR PHAGES AS THERAPEUTIC AGENTS 27 Patricia Veiga-Crespo and Tomas G. Villa

3 ENZYBIOTICS AS SELECTIVE KILLERS OF TARGET 59 Juan C. Alonso and Marcelo E. Tolmasky

4 PHYLOGENY OF ENZYBIOTICS 75 Patricia Veiga-Crespo and Tomas G. Villa

5 : THE ULTIMATE ENZYBIOTIC 107 Vincent A. Fischetti

6 BACTERIOPHAGE HOLINS AND THEIR MEMBRANE- DISRUPTING ACTIVITY 123 María Gasset

7 ANTI-STAPHYLOCOCCAL LYTIC ENZYMES 149 Jan Borysowski and Andrzej Górski

v vi CONTENTS

8 MEMBRANE-TARGETED ENZYBIOTICS 173 María Gasset

9 DESIGN OF PHAGE COCKTAILS FOR THERAPY FROM A HOST RANGE POINT OF VIEW 199 Lawrence D. Goodridge

10 IDENTIFYING PHAGE LYTIC ENZYMES: PAST, PRESENT, AND FUTURE 219 Jonathan E. Schmitz, Raymond Schuch, and Vincent A. Fischetti

11 USE OF GENETICALLY MODIFIED PHAGES TO DELIVER SUICIDAL GENES TO TARGET BACTERIA 253 Lawrence D. Goodridge

CONCLUDING REMARKS: THE FUTURE OF ENZYBIOTICS 269 Patricia Veiga-Crespo and Tomas G. Villa

INDEX 279 PREFACE

The writing of a preface on an old - yet - new subject, such as the one in this book, is always a diffi cult task. It is common knowledge that the discovery of , and subsequently the rest of the antibiotics, has probably been one of the most important scientifi c contributions to civilization. By saving millions of lives, antibiotics automatically increased the half - life of mankind, thus allowing scientists to give their best to society for 20 to 30 additional years. It is also common knowledge that resistance to antibiotics is a con- stant possibility and unfortunately something to be considered every time a new antibiotic goes on the market. Because of this, and because the discovery and design of new antibiotics becomes more and more diffi cult every year, society, through the work of several worldwide research groups, is looking into the use of what one of us (Dr. Vincent Fischetti) has termed “ enzybiotics ” (the result of blending the words “ enzymes ” and “ antibiotics ” ), for treating bacterial and fungal dis- eases, either alone or in combination with antibiotics. The book starts with four chapters in which the potential, advan- tages, and phylogeny of enzybiotics are reviewed. Then, the new ways of controlling infections by Gram- negative bacteria and an updated view of bacteriophage holins are presented. After a review of anti- staphylococcal lytic enzymes, the book goes on to discuss membrane - targeted enzybiotics, as well as the design of phage cocktails for current therapy. Finally, the last two chapters deal respectively with the novel methods to identify new enzybiotics and the use of modifi ed phages to induce suicide in bacteria. All in all, the contributors are all active researchers, involved in the topic of enzybiotics. It is hoped that the joining of different points of view, such as those refl ected in this book, will help to clarify the vii viii PREFACE emerging fi eld of enzybiotics and to consolidate the idea that the thera- pies mediated by these compounds may contribute to the relief of pain and to the control of contagious diseases.

Santiago de Compostela, Spain T omas G. Villa December 24, 2008 P atricia Veiga - Crespo CONTRIBUTORS

Juan C. Alonso Department of Microbial Biotechnology, Centro Nacional de Biotecnolog í a, CSIC, 28049 Madrid, Spain; jcalonso@cnb. csic.es or [email protected]

Jan Borysowski Department of Clinical Immunology, Transplantation Institute, Warsaw Medical University, Poland; [email protected]

Vincent A. Fischetti Professor and Head Laboratory of Bacterial Pathogenesis, Rockefeller University, 1230 York Avenue, New York, NY 10021, USA; [email protected] OR [email protected]

Mar í a Gasset Instituto de Qu í mica - F í sica “ Rocasolano, ” Consejo Superior de Investigaciones Cientí fi cas, Serrano 119, 28006, Madrid, Spain; [email protected]

Lawrence Goodridge Department of Animal Sciences, Colorado State University, Fort Collins, CO 80523, USA; Lawrence.Goodridge@ ColoState.edu

Andrzej G ó rski Department of Clinical Immunology, Transplanta- tion Institute, Warsaw Medical University, and Laboratory of Bacteriophages, L. Hirszfeld Institute of Immunology and Experimental Therapy, Wroc ł aw, Poland; [email protected]

Jonathan E. Schmitz Laboratory of Bacterial Pathogenesis and Immunology, The Rockefeller University, 1230 York Avenue, Box 172, New York, NY 10065, USA; [email protected]

Raymond Schuch Laboratory of Bacterial Pathogenesis and Immunology, The Rockefeller University, 1230 York Avenue, Box 172, New York, NY 10065, USA ix x CONTRIBUTORS

Marcelo E. Tolmasky Center for Applied Biotechnology Studies, Department of Biological Science, College of Natural Science and Mathematics, California State University Fullerton, Fullerton, CA 92834 - 6850, USA; [email protected]

Patricia Veiga - Crespo Department of Microbiology, Faculty of Pharmacy, University of Santiago de Compostela, Spain; patricia. [email protected]

Tomas G. Villa Department of Microbiology, Faculty of Pharmacy, University of Santiago de Compostela, and School of Biotechnology, University of Santiago de Compostela, Spain; [email protected] CHAPTER 1

ENZYBIOTICS AND THEIR POTENTIAL APPLICATIONS IN MEDICINE

JAN BORYSOWSKI1 and ANDRZEJ GÓRSKI1,2 1Department of Clinical Immunology, Transplantation Institute, Warsaw Medical University, Poland 2Laboratory of Bacteriophages, L. Hirszfeld Institute of Immunology and Experimental Therapy, Wrocław, Poland

1. INTRODUCTION

Over the last decade, a dramatic increase in the prevalence of antibiotic resistance has been noted in several medically signifi cant bacterial species, especially Pseudomonas aeruginosa , Acinetobacter baumanii , Klebsiella pneumoniae , as well as , coagulase - negative staphylococci, enterococci, and pneumoniae (Hawkey 2008 ). This unfavorable situation is further aggravated by a shortage of new classes of antibiotics with novel modes of action that are essential to contain the spread of antibiotic - resistant (Livermore 2004 ). In fact, some infectious disease experts have expressed concerns that we are returning to the pre - antibiotic era (Larson 2007 ). Therefore, there is an urgent need to develop novel antibacterial agents to eliminate multidrug - resistant bacteria (Breithaupt 1999 ). A very interesting class of novel (at least in terms of their formal clinical use) antibacterials are enzybiotics. The term “ enzybiotic ” was used for the fi rst time in a paper by Nelson et al. (2001 ) to designate bacteriophage enzymes endowed with bacterial - degrading capacity that could be used as antibacterial agents. While some authors suggest that this name should refer to all enzymes exhibiting antibacterial and even antifungal activity (Veiga- Crespo et al. 2007 ), in this chapter we will discuss only bacterial cell wall - degrading

Enzybiotics: Antibiotic Enzymes as Drugs and Therapeutics. Edited by Tomas G. Villa and Patricia Veiga-Crespo Copyright © 2010 John Wiley & Sons, Inc. 1 2 ENZYBIOTICS AND THEIR POTENTIAL APPLICATIONS IN MEDICINE enzymes (regardless of their source). Other names that are used with respect to enzybiotics are lytic enzymes and hydrolases. The latter refers to the major mode of action of enzybiotics, that is, the enzymatic cleavage of peptidoglycan covalent bonds, which results in the hypotonic of a bacterial cell. Peptidoglycan hydrolases consti- tute an abundant class of enzymes and may be obtained from different sources, for instance, bacteriophages (lysins) and bacteria themselves (bacteriocins and autolysins). Yet another example of well - known enzy- biotics are lysozymes, including hen egg white lysozyme and human lysozyme (a list of representative enzybiotics is shown in Table 1.1 ). In view of the ever - increasing antibiotic resistance of bacteria, the most important characteristics of enzybiotics are a novel mode of anti- bacterial action, different from those typical of antibiotics, and the capacity to kill antibiotic- resistant bacteria (Borysowski et al. 2006 ). Another signifi cant feature of some lytic enzymes is the low probability of developing bacterial resistance (in some cases, the development of enzybiotic resistance results in a reduction in bacterial fi tness and viru- lence; Kusuma et al. 2007 ). The goal of this chapter is to discuss the major groups of enzybiotics, including lysins, bacteriocins, autolysins, and lysozymes, in the context of their potential medical applications.

2. LYSINS

2.1. General Features Lysins or endolysins are double- stranded DNA bacteriophage- encoded enzymes that cleave covalent bonds in peptidoglycan (Borysowski et al. 2006 ; Fischetti 2008 ). They are naturally produced in phage- infected bacterial cells during the course of lytic cycle. At the last stage of the cycle, endolysin molecules degrade peptidoglycan, thereby causing lysis of the bacterial cell and ensuring the release of progeny virions (Young et al. 2000 ). The term “ endolysin ” was introduced to the scientifi c literature by F. Jacob and C. R. Fuerst to stress that molecules act on peptidoglycan from within the bacterial cell in which they are synthesized (Jacob and Fuerst 1958 ). In view of this, it appears that recombinant enzymes acting on the cell wall from outside the cell (e.g., those used for therapeutic purposes) should be referred to as lysins rather than endolysins. Still another name pro- posed to designate a is “ virolysin, ” which is intended to point out the viral origin of these enzymes (Parisien et al. 2008 ). However, this name has not gained popularity and is used very rarely. TABLE 1.1. A List of Representative Enzybiotics Enzybiotic Enzybiotic Source Enzymatic Antibacterial Range Reference Name Class Specifi city PlyC Lysin Phage C1 Amidase S. pyogenes , groups C and E Nelson et al. 2006 streptococci Pal Lysin Phage Dp - 1 Amidase S. pneumoniae Loeffl er et al. 2001 Cpl - 1 Lysin Phage Cp - 1 Muramidase S. pneumoniae Loeffl er et al. 2003 PlyGBS Lysin Phage NCTC Endopeptidase S. agalactiae , groups A, C, G, L Cheng et al. 2005 11261 muramidase streptococci Phage B30 Lysin Phage B30 Endopeptidase S. agalactiae , groups A, B, C, E, Baker et al. 2006 lysin muramidase G streptococci, E. faecalis LambdaSa1 Lysin Prophage Endopeptidase ? Pritchard et al. 2007 prophage LambdaSa1 lysine LambdaSa2 Lysin Prophage Endopeptidase S. pyogenes, S. dysgalactiae , Pritchard et al. 2007 prophage LambdaSa2 glucosaminidase group E streptococci, S. equi , lysine group G streptococci, S. agalactiae PlyG Lysin Phage γ Amidase B. anthracis Schuch et al. 2002 PlyL Lysin Prophage Amidase B. cereus Low et al. 2005 λ Ba02 B. anthracis PlyPH ? ? ? B. anthracis Yoong et al. 2006 PlyB Lysin Phage BcpI Muramidase B. anthracis Porter et al. 2007 Ply118 Lysin Phage A118 Peptidase Listeria Loessner et al. 2002 Ply500 Lysin Phage A500 Peptidase Listeria Loessner et al. 2002 3